Transcriptional regulation is responsible for the strict temporal program of human adenovirus type 2 (Ad2) gene expression characteristic of productive infection. The immediate early E1A 289R protein is required for efficient transcription from all other promoters expressed prior to the onset of viral DNA replication A second transcriptional switch occurs with the onset of the late phase of adenovirus infection and includes activation of a group of late promoters. We have begun to define the cis-acting elements and trans-acting factors required for transcription from one such promoter, that of the IVa2 gene. I propose to continue genetic and biochemical analysis of this promoter, which possesses a downstream TATA element recognized by TFIID and a crucial initiator element. A major aim will be the identification of the initiator-recognizing factor and other factors required for efficient IVa2 transcription, to permit investigation of the mechanism of initiation from an atypical RNA polymerase II promoter. The mechanism that restricts IVa2 transcription to the late phase of adenovirus infection will also be investigated. A sequence-specific factor, termed PuF, which plays an important role in human c-myc transcription, was also identified in studies of the IVa2 promote. This factor will be purified and cloned to permit its detailed characterization, with particular emphasis on the question of whether PuF recognizes primary DNA sequence information or an unusual non-B conformation of the polypurine-polypyrimidine element to which it binds.